Este trabalho é financiado pelo NIH / NCI conceder R01CA-118916

1. Extrair células-tronco epiteliais da pele humana <ol><li> Antes de iniciar o procedimento de isolar células-tronco epiteliais é preciso preparar os respectivos meios e reagentes (ver tabela 1). </li><li> Frescos adulto pele do couro cabeludo humano de procedimentos facelift ou biópsia com punch é coletado e, em seguida incubar em DMEM FBS / 10% / Dispase (4 mg / mL) durante a noite a 4 ° C. Incubação por 2-4 horas a 37 ° C também é eficaz. Pedaços de pele deve ser uma largura máxima de 1 cm para permitir a enzima para penetrar. </li><li> Transferir a pele em uma placa de Petri esterilizada, retire cada cabelo da pele, segurando o eixo do cabelo perto da superfície da pele e puxando com firmeza e sem sobressaltos. Selecione folículos na fase telógena com base na sua morfologia em um microscópio de dissecação, cortar a região protuberância (Figura 1A) folículos transferência para um tubo de 15 mL esterilizados. Folículos anágenos também pode ser usado se cortado em cima 1 / 3 do folículo para obter folículo piloso &quot;protuberância&quot; região (Figura 1B). </li><li> Incubar os fragmentos folículo isolado em uma mistura de 0,05% de tripsina-EDTA (GIBCO) e Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) para 15-20 minutos em temperatura ambiente com agitação periodicamente, adicionar 4 mL DMEM + 10 FBS% para parar a reação, spin baixo por 5 min a 800 rpm. Alternativamente, explantes folículo fragmento de tecido podem ser colocados em cultura sem tripsina digerir a cultura de células de folículos único. </li><li> Elimine o sobrenadante com cuidado (salvo ~ mL 0,2-0,5 para evitar a perda de células) e re-suspensão em 1 mL KCM (médio queratinócitos), sem EGF. </li></ol> 2. Cultura primária epitelial Células-Tronco <ol><li> Antes de cultivar as células-tronco epiteliais, mitomicina C-tratados 3T3-J2 células (células de alimentação) deve ser preparado da seguinte forma. 3T3-J2 células são cultivadas em DMEM com 10% de FBS antes do tratamento. Remova a mídia de cultura de células e lavar duas vezes com PBS, tratar células com mitomicina C 15μg / mL em DMEM sem soro para 2 hr a 37 ° C, 5% CO 2. Remova a mitomicina C contendo DMEM e lavar duas vezes com PBS, as células estão prontas para uso. (Mitomicina C tratados com células 3T3-J2 também pode ser preparado previamente e armazenadas a -80 ° C. Descongele e semente as células um dia antes de cultivo primário de células-tronco epiteliais, incubar a 37 ° C CO, 5% 2. 150.000-200.000 3T3-J2 células por poço da placa 6-bem é recomendado. </li><li> Sementes isoladas células-tronco do folículo de cabelo a partir do passo 1.5 em mitomicina C-tratados 3T3-J2 células em KCM sem EGF durante a noite a 37 ° C, 5% CO 2, alterado para EGF contendo KCM no dia seguinte. As células são cultivadas a 37 ° C em uma atmosfera úmida contendo 5% de CO 2. Todos os queratinócitos são alimentados com KCM contendo EGF a cada 2 dias e cultivadas por 14-20 dias. Verifique celular todos os dias sob o microscópio. Células-tronco do folículo do cabelo irá formar colônias (Figura 2A). Explantes folículo piloso formarão outgrowths após 10-14 dias (Figura 2B). </li></ol> 3. Células-tronco epiteliais passagem <ol><li> Lavar as células com PBS uma vez. Remover as células 3T3-J2 alimentador por Versene tratamento (pré-aquecido a RT). Incubar a cultura dished em Versene por 5 minutos, em seguida, agite e aspirar as células do alimentador. </li><li> Lave as células-tronco do folículo de cabelo com qualquer Versene ou tempo PBS um. </li><li> Adicionar pré-aquecido (a 37 ° C, crítico) de tripsina-EDTA (2X) e incubar a 37 ° C por 7 minutos ou mais se necessário para a célula-tronco de folículo piloso para separar. Não mais que 15 minutos é o melhor. </li><li> Adicionar KCM w / o EGF para parar a tripsina, gentilmente pipeta cima e para baixo para dispersar as células, spin baixo em 200g, a 5 minutos. </li><li> Re-suspender as células com KCM sem EGF Count, e re-placa em mitomicina C tratados com camada de células 3T3-J2. </li></ol> 4. Imortalizar Células-Tronco Epiteliais Pilhas, células-tronco adultas, mesmo, chegar a senescência após a passagem de série e meses em cultura. Imortalização de células-tronco primário é uma forma eficaz para aliviar este problema. <ol><li> Placa primária de células-tronco epiteliais (~ 350 mil células / poço de 6 de placa bem) em uma passagem sobre a camada de alimentação (3T3-J2) e cultura para 2 dias. </li><li> Cultura PA317 LXSN linha celular 16E6E7 um dia após a semeadura primária de células-tronco epiteliais com DMEM contendo 10% FBS. (Esta linha produz o LXSN16E6E7 retrovírus amphotropic que codifica o HPV16 E6 e E7 quadros de leitura aberta, e que pode ser usado para infectar e se estavelmente imortalizar muitos tipos de células). </li><li> Tratar PA317 LXSN linha celular 16E6E7 com mitomicina C por 2 horas (mesmo protocolo com 3T3-J2 células). Adicionar mitomicina C tratados PA317 LXSN linha celular 16E6E7 ao primário de células-tronco epiteliais, co-cultura por 6 dias no KCM com EGF. Renovar KCM fresco com EGF media a cada 2 dias. </li><li> Remover 3T3-J2 e PA317 LXSN 16E6E7 célula com Versene. Adicionar mitomicina-C trataram células 3T3-J2 NHP (neomicina, higromicina, puromicina resistentes, ~ 200.000 células por poço) para as células-tronco epiteliais. Células estão selecionadas em 0,2 mg / ml de G418 (Gibco) para adicionaritional 6 dias. Renovar KCM G418 fresco contendo com EGF media a cada 2 dias. Sobrevivendo células-tronco epiteliais será imortalizado células-tronco. Um bem de células-tronco epiteliais primários que não são co-cultivados com PA317 LXSN 16E6E7 célula deve ser definido como um controlo para a selecção G418, todas as células desse bem deve ser morto por G418 após 6 dias de seleção. </li></ol> 5. Resultados representante Passagem precoce de células epiteliais da pele-tronco e células-tronco epiteliais imortalizada formam colónias apertado consistindo de pequenas queratinócitos (Figura 2A) rodeado por células alimentadoras. Se cultivadas em soro de mídia livre com suplementos definido, sem uma camada de alimentação, as células-tronco se dispersa e não formam colônias apertado (elas crescem como células únicas e pequenos aglomerados). Células-tronco epiteliais imortalizada manter um fenótipo estável por&gt; 12 meses de passagem contínua, mas tendem a formar colônias mais apertado do que as células primárias. Eles não formam colônias de ancoragem independente em ensaios agar suave, indicando que estas células imortalizadas não possuem as características de células cancerosas. Primário e as células-tronco epiteliais imortalizada expressar folículo piloso marcador de células-tronco citoqueratina 15 (Figura 3A), e são capazes de se diferenciar em folículo piloso epidérmica, e linhagens sebáceas (Figura 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figura 1" /> Figura 1. Dedilhados cabelos. Após arrancar a partir de dispase tratados com pele, folículos pilosos aparecem tanto como os cabelos telógeno clube (A) com uma bola de células em torno do fundo do cabelo, ou cabelos anágenos (B) com uma manga de epitélio em torno do comprimento do cabelo. A região bojo telógeno forma uma região distinta morfológicas para micro-dissecção, enquanto o bojo anágena é menos distintas. A lâmpada contém anagen queratinócitos da matriz, o que representa trânsito ampliando-células que também podem ser isoladas e cultivadas. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figura 2" /> Figura células 2. Culturas Stem. (A) células-tronco epiteliais da pele forma apertada colônias epiteliais (designado por E), quando cultivadas em KCM com uma camada de células de alimentação (designados por F). (B) explantes follicle do cabelo dar origem a outgrowths epiteliais. A região bojo telógeno (designada por T) é anexado ao prato e cercada de colônias de células epiteliais com células alimentadoras. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figura 3" /> Figura 3. Diferenciação de células-tronco. (A) As células-tronco manter colônias expressão de marcadores epiteliais como citoqueratina 15. (B) células-tronco epiteliais de pele pode ser diferenciada ao longo da linhagem folículo piloso como determinado pela expressão K6Hf. (C) As células podem ser cultivadas em de-epidermalized derme ou outras matrizes na interface ar-líquido e formar uma camada de epiderme estratificada com cornified, camada granular e camada espinhosa. (D) As células-tronco podem ser induzidas ao longo da linhagem sebáceas com Oil Red glóbulos positivo.

<ol>
	<li>Jones, P. H., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+human+epidermal+stem+cells+from+transit+amplifying+cells+on+the+basis+of+differences+in+integrin+function+and+expression.">Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.</a> Cell. 73, 713-724 (1993).</li><li>Lyle, S., Christofidou-Solomidou, M., Liu, Y., Elder, D. E., Albelda, S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C8/144B+monoclonal+antibody+recognizes+cytokeratin+15+and+defines+the+location+of+human+hair+follicle+stem+cells.">The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells.</a> J. Cell. Sci. 111, 3179-3188 (1998).</li><li>Bac, S. P., Reneha, A. G., Potte, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+the+intestinal+stem+cell+as+a+paradigm.">Stem cells: the intestinal stem cell as a paradigm.</a> Carcinogenesis. 21, 469-476 (2000).</li><li>Slac, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+epithelial+tissues.">Stem cells in epithelial tissues.</a> Science. 287, 1431-1433 (2000).</li><li>It, M., Li, Y., Yan, Z., Nguye, J., Lian, F., Morri, R. J., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+the+hair+follicle+bulge+contribute+to+wound+repair+but+not+to+homeostasis+of+the+epidermis.">Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.</a> Nat Med. 11, 1351-134 (2005).</li><li>Spradlin, A., Drummond-Barbos, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Taylo, G., Lehre, M. S., Jense, P. J., Su, T. T., Lavke, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+follicular+stem+cells+in+forming+not+only+the+follicle+but+also+the+epidermis.">Involvement of follicular stem cells in forming not only the follicle but also the epidermis.</a> Cell. 102, 451-461 (2000).</li><li>Oshim, H., Rocha, A., Kedzi, C., Kobayash, K., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+renewal+of+hair+follicles+from+adult+multipotent+stem+cells.">Morphogenesis and renewal of hair follicles from adult multipotent stem cells.</a> Cell. 104, 233-245 (2001).</li><li>Morri, R. J., Li, Y., Marle, L., Yan, Z., Trempu, C., L, S., Li, J. S., Sawick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotsarelis+G+Capturing+and+profiling+adult+hair+follicle+stem+cells.">Cotsarelis G Capturing and profiling adult hair follicle stem cells.</a> Nat. Biotechnol. 22, 411-417 (2004).</li><li>Ro, C., Roch, M., Gu, Z., Photopoulo, C., Ta, Q., Lyle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In vitro Cell. &#38; Dev. Biol. 44, 236-244 (2008).</li><li>Ohyama, M., Vogel, J. C. G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=delivery+to+the+hair+follicle.">delivery to the hair follicle.</a> J Investig Dermatol Symp Proc. 8, 204-206 (2003).</li><li>Sugiyama-Nakagiri, Y., Akiyama, M., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+follicle+stem+cell-targeted+gene+transfer+and+reconstitution+system.">Hair follicle stem cell-targeted gene transfer and reconstitution system.</a> Gene Ther. 13, 732-737 (2006).</li><li>Stenn, K. S., Cotsarelis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+the+hair+follicle:+fringe+benefits+of+stem+cell+technology.">Bioengineering the hair follicle: fringe benefits of stem cell technology.</a> Curr Opin Biotechnol. 16, 493-497 (2005).</li><li>Hoeller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+rapid+method+to+construct+skin+equivalents+from+human+hair+follicles+and+fibroblasts.+Exp+Dermatol+10.">An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts. Exp Dermatol 10.</a> , 264-271 (2001).</li><li>Navsaria, H. A., Ojeh, N. O., Moiemen, N., Griffiths, M. A., Frame, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reepithelialization+of+a+full-thickness+burn+from+stem+cells+of+hair+follicles+micrografted+into+a+tissue-engineered+dermal+template+(Integra).">Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (Integra).</a> Plast Reconstr Surg. 113, 978-981 (2004).</li><li>Werni, M., Meissne, A., Forema, R., Brambrin, T., K, M., Hochedlinge, K., Bernstei, B. E., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Par, I. H., Zha, R., Wes, J. A., Yabuuch, A., Hu, H., Inc, T. A., Lero, P. H., Lensc, M. W., Dale, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li><li>Kazantsev, A., Goltso, A., Zinchenk, R., Grigorenk, A. P., Abrukov, A. V., Moliak, Y. K., Kirillo, A. G., Gu, Z., Lyl, S., Ginte, E. K., Rogae, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hair+growth+deficiency+is+linked+to+a+genetic+defect+in+the+phospholipase+gene+LIPH.">Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH.</a> Science. 314, 982-985 (2006).</li><li>Tola, J., Ishida-Yamamot, A., Riddl, M., McElmurr, R. T., Osbor, M., Xi, L., Lun, T., Slatter, C., Uitt, J., Christian, A. M., Wagne, J. E., Blaza, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+epidermolysis+bullosa+by+transfer+of+wild-type+bone+marrow+cells.">Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.</a> Blood. 113, 1167-1174 (2009).</li><li>Wagne, J. E., Ishida-Yamamot, A., McGrat, J. A., Hordinsk, M., Keen, D. R., Riddl, M. J., Osbor, M. J., Lun, T., Dola, M., Blaza, B. R., Tolar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+transplantation+for+recessive+dystrophic+epidermolysis+bullosa.">Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.</a> N Engl J Med. 363, 629-639 (2010).</li><li>Muraue, E. M., Gach, Y., Grat, I. K., Klausegge, A., Mus, W., Grube, C., Meneguzz, G., Hintne, H., Baue, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Correction+of+Type+VII+Collagen+Expression+in+Dystrophic+Epidermolysis+Bullosa.">Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa.</a> J Invest Dermatol. , (2010).</li><li>Y, H., Kuma, S. M., Kossenko, A. V., Show, L., X, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J Invest Dermatol. 130, 1227-1236 (2010).</li><li>Nishimur, E. K., Grante, S. R., Fishe, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+graying:+incomplete+melanocyte+stem+cell+maintenance+in+the+niche.">Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche.</a> Science. 307, 720-724 (2005).</li></ol>

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Os métodos de extração de células e cultura descritos são surpreendentemente fáceis e reprodutíveis. Temos gerado culturas de células-tronco epiteliais de dezenas de indivíduos através de uma faixa etária ampla, incluindo pacientes com defeitos da pele herdado 18. É melhor para começar o processo no dia da colheita de tecidos, no entanto células permanecerá viável nos meios de comunicação sobre o gelo por vários dias, facilitando o transporte de noite, se necessário. Pele facelift descarta...

Media dos queratinócitos (KCM) [DMEM e Ham s F12 (GIBCO, 3:1), adenina (Sigma, 180 mM), 10% de soro fetal bovino (GIBCO), a toxina do cólera (ICN, 0,1 nM), a penicilina / estreptomicina (GIBCO, 100 U / ml e 100 mg / ml, respectivamente), hidrocortisona (Sigma, 0,4 mg / ml, 1,1 mM), T/T3 (transferrina, GIBCO, 5 mg / ml, 649 nM e triiodo-l-thyronine, Sigma, 2 nM) , a insulina (Sigma, 5 mg / ml, 862 nM) e EGF (Sigma, 10 ng / ml, 1,6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

A homeostase de todos os tecidos auto-renovação é dependente das células-tronco adultas. Como células-tronco indiferenciadas sofrer divisões assimétricas, que geram células-filhas que mantêm o fenótipo de células-tronco e trânsito ampliando-cells (células TA) que migram do nicho de células-tronco, são submetidos a rápida proliferação e diferenciação terminal para repovoar o tecido. Células-tronco epiteliais foram identificados na epiderme, folículo piloso, e intestino como células com alto potencial proliferativo in vitro e como o ciclismo slow-label de retenção de células in vivo 1-3. Adulto, tecido-específicos de células-tronco são responsáveis ​​pela regeneração dos tecidos em que residem durante turnover fisiológico normal, assim como em épocas de estresse 4-5. Além disso, as células-tronco são geralmente consideradas como multi-potentes, possuindo a capacidade de dar origem a vários tipos de células dentro do tecido 6. Por exemplo, roedor cabelos células-tronco do folículo pode gerar epiderme, glândulas sebáceas e folículos pilosos 7-9. Nós mostramos que as células-tronco do humano região bojo folículo piloso apresentam potencialidade multi-10. Células-tronco tornaram-se uma ferramenta valiosa na pesquisa biomédica, devido à sua utilidade como um sistema in vitro para estudar a biologia do desenvolvimento, diferenciação, tumorigênese e para a sua eventual utilidade terapêutica. É provável que células-tronco adultas do epitélio será útil no tratamento de doenças como displasias ectodérmicas, monilethrix, síndrome de Netherton, doença de Menkes, epidermólise bolhosa hereditária e alopecias 11-13. Além disso, outros problemas de pele, como queimaduras, feridas crônicas e úlceras irá beneficiar de terapias com células-tronco relacionadas 14,15. Dado o potencial para a reprogramação de células adultas em estado pluripotentes (células iPS) 16,17, as células adultas de fácil acesso e expansível-tronco na pele humana pode fornecer uma valiosa fonte de células para a indução e terapia a jusante de uma ampla gama de doenças, incluindo diabetes e doença de Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Uma maneira rápida e robusta de isolar células-tronco adultas viável epiteliais da pele humana é descrito. O método utiliza a digestão enzimática da matriz de colágeno da pele, seguido por arrancar dos folículos pilosos e isolamento de células em suspensão simples ou fragmentos de tecido para cultura de células.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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28.2K visualizações.  University of Massachusetts Medical School. Uma maneira rápida e robusta de isolar células-tronco adultas viável epiteliais da pele humana é descrito. O método utiliza a digestão enzimática da matriz de colágeno da pele, seguido por arrancar dos folículos pilosos e isolamento de células em suspensão simples ou fragmentos de tecido para cultura de células.

Vídeo: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Biologia

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolamento de células-tronco de xenoenxertos Humano do Câncer de pâncreas

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolamento manual de células-tronco adiposo-derivadas de Lipoaspirates Humanos

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, cultura e transplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...